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Histology 1.2. Immunohistochemistry
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• Immunohistochemistry uses the principle of
  immunity
• During development the immune system recognizes
  foreign
• proteins as antigens
• If foreign proteins invade the body, this evokes
  immune response
• One type of immune response is the production of
  highly specific
• molecules against the foreign proteins. These
  are called antibodies,
• binding with high affinity to the antigens
• Immunocytochemistry utilizes these antibodies for
  the localization
• of tissue components
• Production of antibodies
• A tissue constituent is extracted from the body
  of X animal (e.g. goat),
• and purified
• 2. This material is injected into the bloodstream
  of Y animal (e.g. rabbit),
• where it behaves as antigen and evokes immune
  response,
• thus, production of highly specific antibodies
• 3. The antibody can be extracted from the blood
  of Y animal, purified and
• characterized.

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• Preparation of tissues for immunohistochemistry
• Collection of samples (tissue blocks from
  experimental
• animals, biopsy, smears, etc.)
• Fixation - immersion (drop the tissue block into
  fixative)
• - perfusion through the heart

• Perfusion
• Deep anaesthesia (Nembutal, etc.)
• Cannule introduced to the left ventricle
• or into the aorta
• Wash out the blood with a saline
• Fix with paraformaldehyde and/or
• glutaraldehyde
• 5. Removal of the wanted tissue or organ
• immersion-fixed for some hours
• 6. Sectioning
• 7. Incubation of sections

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• An example pre-embedding
• immunohistochemical reaction
• Antigen (green triangle)-antibody
• binding in the tissue
• 2. Antigen-antibody binding
• between the primary antibody
• and the secondary antibody
• labelled with either a gold
• particle, or a fluorescent dye,
• or an enzyme catalysing
• a chromogen reaction

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The results
Epithelial cells infected with influensa viruses
(brown dots) in the wall of a bronchus in the
lung
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Nerve cells containing the enzyme nitrogen
monoxide synthase (DAB reaction, brown
precipitate)
Endothelial cell culture Red fluorescence actin
cytoskeleton Green fluorescence tubulin Blue
DAPI staining of the nucleus (not immune
staining)
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The electron microscope
Brief history 1920 physicists discovered that
accelerated electrons behave in vacuum jut like
light - they travel in straight lines and their
wavelength is about 100.000 times smaller
than that of light. - the electron beam can be
manipulated with electromagnetic field just
like the light with glass lenses 1931 Ernst
Ruska built the first electron microscope
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The transmission electron microscope (TEM)
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Electron source triode gun 1. filament
tungsten, heated up to 2700oC emits electron
cloud 2. Wehnelt cylinder bunches the electrons
into finely focused point 3. anode has a hole in
it so that the accelerated electron beam get
through it with a speed of several 100.000
km/sec Magnification with the help of
electromagnetic lenses changing the strength
of the current within the coils changes the
magnification Image formation the focussed
electron beam reaches the extremely thin
specimen (60-90 nm), passes through it and the
image is projected to a fluorescent screen the
specimen has to be treated with heavy metal salts
in order to get contrasty image
(stainingcontrasting)
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• Preparation fo tissues for electron microscopy
• Fixation buffered solutions of paraformaldehyde
  and
• glutaraldehyde (immersion and perfusion)
• 2. Staining/contrasting with osmium tetroxide
• 3. Dehydration in ascending series of ethanol
  (50-100)
• Staining/contrasting with 70 ethanol saturated
  with uranyl acetate
• 4. Intermediate solvent propylene oxide
• 5. Embedding in synthetic resins e.g. Durcupan
  ACM (liquid at room
• temperature, polymerises at 56 oC)
• 6. Preparation of semithin (0.5 mm) and ultrathin
  (60-90 nm) sections
• Staining/contrasting with lead citrate.

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The ultramicrotom
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The electron micrograph
nucleus
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Scanning electron microscope (SEM)

• Suitable to observe the surface of tissue
  components
• Parts of SEM
• Electron optical column (short with 3 lenses)
• Specimen chamber
• Works like the tv screen
• - The electron beam hits the surface of the
  specimen which
• has to be covered with a thin layer of metal
  (e.g. gold)
• Secondary electrons are detected and turned into
  an electrical
• signal.
• In the monitor electrical signal is turned into
  light to produce
• an image.

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SEM images
Red and white blood cells
Blood clotting
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Pre-embedding immunocytochemistry at electron
microscopic level Its steps are similar to
those of light microscopic ICC but - Triton
X-100 detergent is not allowed to use - Instead
Triton X-100 freeze-thaw in liquid nitrogen
helps the penetration of antibodies - The
immunoreaction is carried out on 60-80 mm
vibratome sections
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• Further steps after the immunoreaction
• contrasting buffered 1 OsO4 30-60 min
• Dehydration in ascending series of ethanol 10-10
  min
• (70 ethanol is saturated with uranyl acetate)
• Intermedier solvent propylene oxide 10 min
• Durcupan propylene oxide 11 30 min
• - Durcupan resin overnight
• - Mounting on glass slide in Durcupan resin
• - Polimerization 56 oC-on one day
• - re-embedding for ultrathin sectioning
• - Preparation of ultrathin sections (60-90 nm)
  in ultramicrotome
• - Contrasting with lead citrate 2-10 min
• - View in electron microscope

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Light microscopic level Electron microscopic
level
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Postembedding immunogold labelling - Carried out
on ultrathin sections - Secondary antibody is
decorated with a colloidal gold particle
Localization of gonadotrop hormon presynaptic
membrane protein